Impact of Drying Method on Antioxidant, Anti-Diabetic, and Anti-Proliferation Activities of Cirsium Setidens in Vitro

Acta Alimentaria, Vol. 47 (1), pp. 44–51 (2018) DOI: 10.1556/066.2018.47.1.6 IMPACT OF DRYING METHOD ON ANTIOXIDANT, ANTI-DIABETIC, AND ANTI-PROLIFERATION ACTIVITIES OF CIRSIUM SETIDENS IN VITRO H.F. GUO and M.H. WANG* Department of Medical Biotechnology, College of Biomedical Science, Kangwon National University, Chuncheon, 24341. Republic of Korea (Received: 7 March 2017; accepted: 30 May 2017) This study investigated the infl uences of drying method (oven-, freeze-, and shade-drying) and extraction solvent (ethanol and water) on the bioactivities of Cirsium setidens. Antioxidant activity was evaluated by DPPH radical scavenging ability, anti-diabetic activity was determined by the inhibitory activity of two enzymes: α-glucosidase and α-amylase, while anti-proliferation activity was assessed by MTT assay of three human cancer cell lines (KB, A549, and PC-3). Results indicated that bioactivities were extremely affected by solvent; water extracts contained more phenolics, exhibited strong anti-diabetic effect, but no activity of anti-proliferation, while the ethanolic extracts rich in fl avonoids showed profound DPPH radical scavenging and anti-proliferation ability, yet low activity of anti- diabetes. Among the drying methods, freeze-drying extracts preserved more fl avonoids and exhibited better activity of anti-proliferation, while shade-drying extracts contained higher phenolics and showed stronger activity on anti- diabetes, oven-drying gave the lowest content of phenolics. Hence, antioxidant and anti-diabetic effects were positively related to phenolic content, meanwhile an extremely signifi cant correlation coeffi cient had been found between anti-proliferation activity and fl avonoid content, it can be concluded that drying method and extraction solvent affect bioactivities by phenolic and fl avonoid contents. Keywords: antioxidant, anti-diabetic, anti-proliferation, Cirsium setidens, drying method Cirsium setidens (Asteraceae family), is one of the thistles native in Gangwon do, Republic of Korea. Cirsium setidens is a nutrient rich plant abundance of calcium and vitamins, therefore residents are accustomed to eating leaves and stems of the plant as vegetables. It is also employed as a folk medicine to treat hematemesis, hypertension, and hematuria. Since C. setidens exhibits plenty of benefi ts, it is worthy to explore the use of C. setidens in cosmetics, medical and functional food. Generally, plants are marketed in forms of extracts or powders produced from dried plants. The removal of water can be classifi ed by temperature into three categories: drying at high, normal, or low temperature. The heating process evaporates moisture on the surface and forces the inside moisture to travel to the surface, there it is evaporated and freeze drying sublimates the moisture directly into vapour. Heating accelerated the drying process and prevented reproduction of microorganisms, while at normal temperature the process needs more time and freezing has a higher cost (RAGHAVAN & ORSAT, 2007). However, drying process seriously affected the bioactivities of the product through enzymatic degradation, volatilization, and decomposition (MA et al., 2013). Therefore, a process optimization should be carried out to provide the best nutritional values and functional activity in the fi nal products. According to a study, hot temperature treatment provides more bioactive compounds than freeze-drying treatment in case of olive leaves * To whom correspondence should be addressed. Phone: +82-33-250-6486; fax: +82-33-259-5644; e-mail: [email protected] 0139–3006 © 2018 Akadémiai Kiadó, Budapest GUO & WANG: IMPACT OF DRYING ON CIRSIUM SETIDENS 45 (AHMAD-QASEM et al., 2013). Whereas, CHAN and co-workers (2009) reported that freeze- drying provided a signifi cant increase in total phenolics content, ascorbic acid equivalent antioxidant activity, and reducing power activity opposite to other methods. In this case, whether the drying process of C. setidens affected bioactivity is worthy to be investigated. To our knowledge, there is no literature focusing on this fi eld, and we intend to use oven-, freeze-, and shade-drying methods to dehydrate fresh C. setidens leaves and to compare the bioactivities of their ethanol and water extracts. 1. Materials and methods 1.1. Chemicals and reagents Folin–Ciocalteu reagent, aluminium chloride hexahydrate, tannic acid, quercetin, acarbose, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1,1-diphenyl-2- picrylhydrazyl (DPPH), p-nitrophenyl-α-d-glucopyranoside (pNPG), dinitrosalicylic acid (DNS), α-glucosidase from Saccharomyces cerevisiae, and α-amylase from porcine pancreas were purchased from Sigma (St. Louis, MO, USA). RPMI 1640, DMEM (high glucose), fetal bovine serum (FBS), and trypsin-EDTA were acquired from Hyclone (Thermo Scientifi c, Waltham, MA, USA). The culture supplies were purchased from SPL Brand Products (SPL, Republic of Korea). All chemicals or reagents were of analytical grade. 1.2. Materials Fresh C. setidens leaves were obtained in Gangwon do, Republic of Korea. The fresh plants were divided into three parts and dried by oven-drying, freeze-drying, and shade-drying. Oven-drying was carried out in an electric thermo static drying oven at 55 °C. Freeze-drying was done in a freeze dryer (FDE-0350, Humanlab instrument, Republic of Korea) at –48 °C, and the shade-drying was in a draughty house avoid of sunlight at room temperature (25±2 °C). The dried plants were ground in a rotary mill and extracted by ethanol or water. 1.3. Total phenolic and fl avonoid content Total phenolic content was evaluated following the method reported by SINGH and co-workers (2002) with modifi cation. Sample solution reacted with 10% Folin–Ciocalteu reagent and 7.5% sodium carbonate for 30 min in dark, the absorbance was detected at 750 nm. Tannic acid was chosen as the standard and total phenolics content was expressed as mg tannic acid equivalent/g (mg TAE/ g). Total fl avonoid content was detected by the mixture of aluminium chloride solution and sample ethanol solution. The absorbance of the solution was measured at 405 nm after 1 h. Quercetin was employed as the standard, thus total fl avonoids content was expressed as mg quercetin equivalent/g (mg QE/g). 1.4. DPPH radical scavenging activity DPPH scavenging activity was measured according to the method of ERKAN and co-workers (2008) with a slight modifi cation. Five hundred microlitres of 0.2 mM DPPH solution (in methanol) was mixed with the same volume of sample solution (in methanol) at various Acta Alimentaria 47, 2018 46 GUO & WANG: IMPACT OF DRYING ON CIRSIUM SETIDENS concentrations. It was left to stand for 30 min in darkness at room temperature after vortexing, then the absorbance was measured at 515 nm using a microplate spectrophotometer reader (ELx800TM, BioTek, Winooski, VT, USA). α-Tocopherol was used as positive control. 1.5. Alpha-glucosidase inhibitory assay IM Alpha-glucosidase activity was measured by a modifi ed method described by K and co- workers (2005), 4 mM pNPG substrate solution (pH 6.9) was added to the mixture of extract and α-glucosidase solution after pre-incubation. The reaction was stopped after 20 min incubation at 37 °C by 0.1 M Na2CO3 and measured at 400 nm. Acarbose was used as positive control and the blank was distilled water. 1.6. Alpha-amylase inhibition assay Alpha-amylase inhibition assay was carried out according to MCCUE and SHETTY (2004). After the pre-incubation of the mixture of extract and α-amylase, 2% starch was added, and the mixture was incubated for 5 min. DNS (pH 6.8) was added and the reaction was stopped by heating at 100 °C for 15 min. The absorption was measured at 540 nm (room temperature). Acarbose was employed as positive control and the blank was distilled water. 1.7. Anti-proliferation activity of the cancer cells KB (human oral carcinoma cell), A549 (human lung cancer cell), and PC-3 (human prostate cancer cell) cell lines were purchased from Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) and cultured as the supplier described. The anti-proliferation activity was determined by MTT assay with 24, 48, or 72 h treatment of indicated concentration of extracts, followed by the procedure reported by VAN MEERLOO and co-workers (2011) and calculated by the percentage of untreated control. 1.8. Statistical analysis The results were expressed in terms of mean (n=3) ± standard deviation. Statistical analysis was conducted by SPSS 21 (SPSS Institute, Cary, NC, USA) through one-way analysis of variance (ANOVA followed by Duncan’s multiple-range test) to determine the signifi cant differences between the groups. 2. Results and discussion 2.1. Antioxidant activity DPPH is a free radical commonly employed to measure the antioxidant activity. As shown in Figure 1, shade-drying ethanol extract (SDE) and oven-drying ethanol extract (ODE) almost abolished the free radical completely at 200 μg ml–1, followed by freeze-drying ethanol extract (FDE), oven-drying water extract (ODW), freeze-drying water extract (FDW), and shade-drying water extract (SDW). Among the three drying methods, oven-drying displayed greater activity, shade-drying results varied with extraction solvent, and freeze-drying exhibited less activity in scavenging DPPH free radical. Acta Alimentaria 47, 2018 GUO & WANG: IMPACT OF DRYING ON CIRSIUM SETIDENS 47 Fig.1. DPPH free radical scavenging activity of C. setidens extracts : SDE; : FDE; : ODE; : SDW; : FDW; : ODW; : α-Tocopherol Table 1. Alpha-glucosidase and α-amylase inhibitory

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